qPCR – pCR2.1/RLOv Standard Curves Testing

Earlier today, I created dilution series of the following two linearized plasmids to develop qPCR assays:

  • pCR2.1/RLOv_DNA_helicase
  • pCR2.1/RLOv_head_to_tail

Master mix calcs: 20151106 – qPCR RLOv Standard Curves

All samples were run in triplicate on a CFX96 thermal cycler (BioRad).

Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Data File (CFX96): Sam_2015-11-06 18-17-41_CC009827.pcrd
qPCR Report (PDF): Sam_2015-11-06 18-17-41_CC009827.pdf

DNA Helicase Curve

Amplifcation plots and the standard curve best fit line looks really good. Efficiency is very close to 100% and the R^2 = 0.99. Additionally, virtually all of the replicates are very tight. This looks like it will be totally usable as a standard curve for developing a qPCR assay that targets the RLOv DNA helicase gene.

 

Head-to-tail Curve

This curve is way wonky. Interestingly, the end-point fluroescence levels for this curve 5-fold lower than the DNA helicase curve. I’ll likely repeat this qPCR to see if these crappy results are repeatable. However, having a single qPCR assay (the DNA helicase standard curve) for RLOv detection/quantification might be sufficient, rendering a second qPCR assay unneeded.

Standard Curve Dilutions – pCR2.1/RLOv Plasmids

Set up standard curve dilutions to use for qPCRs with the following two linearized plasmids:

  • pCR2.1/RLOv_DNA_helicase
  • pCR2.1/RLOv_head_to_tail

Used a spreadsheet developed by Nate many moons ago to determine necessary volumes based on plasmid size to obtain desired copy numbers.

Dilutions were made in TE Buffer.

DNA helicase dilutions (Google Sheet): 20151106 – Dilution table RLOv_DNA_helicase_qPCR_Standard_Curve

Head-to-tail dilutions (Google Sheet): 20151106 – Dilution table RLOv_head_to_tail_qPCR_Standard_Curve

Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

PCR – RLOv Clones

Colony PCRs were performed on each of the transformations from 20151015 (RLOv_ DNA_helicase, RLOv_head_to_tail, RLOv_membrane_gene_1, RLOv_membrane_gene_2, RLOv_tail_to_fiber) to confirm successful ligations in plasmid pCR2.1 using the M13F/R vector primers.

Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp100+x-gal plate and then used to inoculate the respective PCR reactions.

Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.

Master mix calcs are here (Google Sheet): 20151019 – Colony PCRs RLOv

Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).

30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.

Results:

 

All the PCRs look good. All white colonies selected contain a PCR product of appropriate size (i.e. larger than the blue colonies; negative [-C] control). Will select clones #1 from each to grow up for plasmid prep.

Cloning – Purified Abalone RLOv PCR Products

Purified PCR products from 20151008 (in situ hybridization [ISH] primers) & 20151009 (qPCR primers) RLOv primers were used for cloning. The qPCR primers are intended to develop a qPCR standard curve and the ISH primers are intended to develop three ISH probes.

  • RLOv_DNA_helicase (for qPCR)
  • RLOv_head_to_tail_gene (for qPCR)
  • RLOv_membrane_gene_1 (for ISH)
  • RLOv_membrane_gene_2 (for ISH)
  • RLOv_tail_fiber_gene (for ISH)

The PCR products were separately ligated using The Original TA Cloning Kit (Invitrogen).

LIGATIONS

REAGENT SINGLE REACTION VOL (μL) x 5.5
Purified PCR 5 NA
10x Ligase Buffer 1 5.5
pCR2.1 Vector 2 5.5
H2O 1 5.5
T4 DNA Ligase 1 5.5
TOTAL 10 22

Ligation reactions were set up on ice.Combined 5μL of purified PCR product with 5μL of ligation master mix in a 1.5mL snap cap tube. Incubated 24hrs @ RT.

 

TRANSFORMATIONS

50μL of X-gal (20mg/mL) was added to a LB-Amp100 plates, spread and warmed @ 37C.

Five vials of OneShot TOP 10 chemically competent cells (Invitrogen) were thawed on ice. 3μL of the ligation reaction was added to the cells, gently mixed and incubated on ice for 5mins. 50μL of cells were transferred to the LB-Amp100+X-gal plates, spread and incubated O/N at 37C.

PCR – RLOv for Cloning & Sequencing

After yesterday’s confirmation that the qPCR primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv, I needed to generate PCR products to clone and sequence.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

All samples were run in duplicate.

Master mix calcs are here: 20151009 – PCR RLOv

Cycling Params (PTC-200; MJ Research)

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 95
  • 600
40 Cycles
  • 95
  • 55
  • 72
  • 15
  • 15
  • 30

Samples were run on a 0.8% agarose 1x TBE gel, stained with ethidium bromide.

Results:

Amplification looks great. No amplification in no template controls (NTCs). Excised bands and purified products using Ultrafree DA Spin Columns (Millipore). Samples will be stored @ 4C until I am able to clone them for sequencing.

 

Gel image showing excised bands. And, it’s a complete hack job, which is embarrassing…

qPCR – RLOv Specificity Check

After yesterday’s confirmation that the primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv (and don’t amplify RLO alone), I needed to confirm that the qPCRs only generated a single product in each reaction via melt curve analysis.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

NOTE: Remaining volume of template DNA wasn’t going to be sufficient for all reactions, so added 100μL of NanoPure H2O. Seeing how early the amplification was in yesterday’s qPCR (Cq ~15), this dilution should be fine.

All samples were run in duplicate.

Master mix calcs are here: 20151009 – qPCR RLOv

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:
qPCR Report (PDF): Sam_2015-10-09 12-36-54_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-09 12-36-54_CC009827.pcrd

Both primer sets amplified a single PCR product. This is demonstrated by the single melt peak for each primer set.

qPCR – New Withering Syndrome Phage Primers

Ran qPCR with the newly designed primers and probes for the following targets:

  • DNA Helicase (RLOv)
  • Head-to-tail gene (RLOv)
  • WSN1 (RLO)

Template DNA used:

In the histology scoring pictures below, the “New” column refers to histology scores for the presence of the phage. A score = 0 means no phage.

  • 06:5-6 (RLO only)

  • 06:6-54 (RLOv)

  • UW08:22-11A (naive pinto abalone; no RLO)

 

Master mix calcs are here: 20151008 – qPCR WS phage

All samples were run in duplicate. Cycling params, plate layout, etc. can be seen in the qPCR Report (see below).

Results:

qPCR Report (PDF): Sam_2015-10-08 17-45-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-08 17-45-38_CC009827.pcrd

ORANGE – WSN1; BLUE – DNA Helicase; GREEN – Head-to-tail

The results look great! The two RLOv (phage) primer sets only amplify in the sample that has histological confirmation of the presence of phage (06:6-54). They do not amplify in the RLO-only (no phage; 06:5-6) sample, demonstrating that these two primer sets are indeed specific to the phage and don’t  amplify the RLO.

The withering syndrome primers (WSN1) were run to confirm that there aredetectable levels of RLO in both the RLOv & RLO samples, to further support the evidence showing the specificity of the two phage primer sets.

Will use the two RLOv primer sets in a conventional PCR for cloning/sequencing and development and validation of a qPCR standard curve.